The long-term goal of the proposed research is to understand, in detail, the role(s) of U1 snRNP in the related processes of exon definition and 5' splice site recognition. Although U1 snRNP was the first snRNP to be extensively characterized, and the first snRNP demonstrated to be required for pre-mRNA splicing, the mechanisms by which it functions remain poorly understood. Preliminary studies show that highly purified U1 snRNP binds to an authentic 5' splice site without the assistance of any cofactors. This binding is quantitative, stable, and highly specific; most remarkably, binding requires -twenty nucleotides of the upstream exon. These findings will be pursued under four aims. Identification and functional characterization of components that comprise an exon definition complex. U1 snRNP nucleates the assembly of a pre-mRNA/protein complex that spans the upstream exon. A new tandem affinity strategy will be used to purify this complex and the identity of its protein constituents will be determined by mass-spec sequence analysis. The functional roles that each component plays in exon definition will be assessed via depletion and functional reconstitution using recombinant proteins. Biochemical characterization of U1 snRNP binding to the 5' splice site: mechanism and substrate requirements. Stable U1 snRNP binding to the 5' splice site requires exonic determinants and the base-pairing region. Those features within the pre-mRNA that promote stable U1 snRNP binding, will be determined using a variety of modification interference approaches including NAIM. These studies will reveal essential interactions between U1 snRNP and the pre-mRNA and will assess the contribution that base pairing makes to binding. Exonic sequences that promote stable Ul snRNP binding to a suboptimal 5' splice site. A randomization selection approach has identified twenty specific exonic variants that promote stable U1 snRNP binding to a "weak" 5' splice site. Binding studies with purified U1 snRNP and an affinity selection approach in HeLa cell extract will be used to determine how the exonic sequences exert their effects. Proximal-distal 5' splice site choice and the role of U1 snRNP. SR proteins and hnRNPA1 have antagonistic effects on 5' splice site choice; SR proteins promote use of proximal sites and hnRNPA1 promotes use of distal sites. These effects may result from modulation of U1 snRNP occupancy of 5' splice sites. These models will be tested using new pre-mRNAs with duplicated 5' splice sites "pre-loaded" with U1 snRNP. In aggregate, the studies proposed in these interrelated aims have a high probability of providing fundamental new insight into the role(s) of U1 snRNP in 5' splice site identification and exon recognition. Furthermore, the results may be relevant to our understanding of regulated alternative splicing.